Recently, the interest for surface sensitive measuring techniques has increased markedly as several so-called label-free optical techniques have been developed for measuring and quantifying biomolecular interactions. A so far frequently used such optical technique is based on surface plasmon resonance, hereinafter often referred to as SPR.
When light travels from an optically denser medium (i.e. having a higher refractive index) to a less dense medium (i.e. having a lower refractive index), total internal reflection (TIR) occurs at the interface between the two media if the angle at which the light meets the interface is above a critical angle. When TIR occurs, an electromagnetic "evanescent wave" propagates away from the interface into the lower refractive index medium.
If the interface is coated with a thin layer of certain conducting materials (e.g. gold or silver), the evanescent wave may couple with free electron constellations, called surface plasmons, at the conductor surface. Such a resonant coupling occurs at a specific angle of the incident light, absorbing the light energy and causing a characteristic drop in the reflected light intensity at that angle. The surface electromagnetic wave creates a second evanescent wave with an enhanced electric field penetrating into the less dense medium.
The resonance angle is sensitive to a number of factors including the wavelength of the incident light and the nature and the thickness of the conducting film. Most importantly, however, the angle depends on the refractive index of the medium into which the evanescent wave of the surface plasmon wave propagates. When other factors are kept constant, the resonance angle is thus a direct measure of the refractive index of the less dense medium, the angle being very sensitive to refractive index changes in the medium. For a detailed description of the dielectric equations describing this dependence it may be referred to Kretschmann, E., Z. Phys. B241, 313 (1971).
The SPR evanescent wave decays exponentially with distance from the interface, and effectively penetrates the lower refractive index medium to a depth of approximately one wavelength. Therefore, only changes in refractive index very close to the interface may be detected.
If the metal film is covered with an appropriate sensing layer (e.g. an antibody) capable of specific interaction with a molecule (e.g. an antigen) present in a fluid sample contacted with the sensing layer, SPR based chemical sensors may be constructed, the interaction at the sensor surface changing the solute concentration and bound surface concentration and thus the refractive index within the penetration range of the evanescent wave. A variety of SPR-based chemical sensors have been developed wherein the change of the reflectance curve (reflected light intensity versus angle of incidence or wavelength) with time is measured, which change is correlated to the surface refractive index.
Different techniques may be used for bringing the light to interact with the sensor surface. A commonly used detection system is based on the Kretschmann configuration (Kretschmann and Raether, Z. Naturforsch. Teil A 23: 2135-2136, 1968). In this configuration a thin layer of the reflective metal (gold or silver) is deposited on the base of a prism and TM polarized, monochromatic light is coupled by the prism to the SPR-wave.
An example of a commercial biosensor system constructed on the basis of the Kretschmann configuration is BIAcore.TM., marketed by Pharmacia Biosensor AB (Uppsala, Sweden). This biosensor system incorporates a Kretschmann configuration-based SPR-detection system with a microfluidic system to control the flow of reagents required in the analyses. With this apparatus, biomolecular interactions occurring at the sensor surface may be monitored in real time. The apparatus and theoretical background thereof has been fully described by Jonsson et al., 1991, BioTechniques 11, 620-627. It is also referred to our U.S. Pat. No. 5,313,264.
When monitoring the biomolecular interactions, the development of the SPR-response, or surface film refractive index, with time is followed. From this relationship not only the biomolecule analyte concentration may then be determined but also kinetic parameters, such as association and dissociation rate constants for the interaction of the biomolecule with the sensor surface.
As an alternative to the reflectance minimum angle, the angle of the centroid of the reflectance curve may be monitored.
Rather than measuring the incident light angle where SPR (i.e. reflectance minimum) occurs, some SPR sensors, as already mentioned above, introduce the light at a fixed angle and measure the wavelength at which SPR takes place (see e.g. U.S. Pat. No. 5,359,681), SPR in both cases, however, representing a reflectance minimum of the reflectance curve.
When studying kinetic parameters, it is, of course, important to know when the interaction at the surface is mass transport limited and when it is reaction-kinetically controlled. This may not readily be determined from the above-mentioned refractive index versus time curve, and there is therefore always a risk of an incorrect estimate of kinetic constants. Further, it may not be readily determined from the refractive index versus time curve whether the interaction at the surface has caused any structural change of the surface resulting in a heterogeneity.
In other contexts than the chemical SPR sensors, parameters such as the intensity of the reflectance minimum (R.sub.min) and the halfwidth (.PHI..sub.1/2) of the reflectance curve have been studied.
Pockrand, I., Surface Science, vol. 72, p. 577-588 (1978) describes the influence of thin dielectric coatings on the properties of surface plasma oscillations propagating along a metal surface using an attenuated total reflection (ATR) arrangement to excite SPR. A general shift and a broadening of the resonance curve are observed. The depth of the resonance R.sub.min is unaffected by transparent coatings, whereas it is strongly dependent on the thickness of an absorbing coating.
Fontana, E., Pantell and Moslehi, Applied Optics, vol. 27, (1988) p. 3336 characterizes dielectric-coated metal mirrors using surface plasmon spectroscopy. Analytical expressions for SPR angle shift, halfwidth and reflectivity minimum are described.
Chu, K. C., et al., Mol. Cryst. Liq. Cryst., vol. 59, p. 97-108, (1980) discloses a study of isotropic-nematic phase transition of 4-cyano-4'-n-pentylbiphenyl on gold as a function of temperature by surface plasmon resonance technique. Changes in reflectance curve halfwidth and the occurrence of two reflective dips were observed. One of the two resonant angles corresponds to the refractive index of the isotropic phase, and the other to the refractive index of the nematic phase. The relationship between the dip depths of the "double dip reflectivity curve" was used as a measure of the fraction of the medium in each of the two phases.
Pollard, J. D., and Sambles, J. R., Optics Communications, vol. 64, p. 529-533 (1987) describes the analysis of the time-dependence of the SPR reflectance curve parameters, reflectance minimum angle, reflectance minimum depth, and reflectance curve width to study the lateral extension of the two phases of a condensed liquid, i.e. as droplets and as a homogeneous film, respectively. The existence of two distinct refractive indices in different parts of an illuminated gold surface gave two reflectance curves which together gave a reflectance curve with two minima, the depth of the respective minimum corresponding to the mutual degree of coverage of the gold surface.
Rothenhausler and Knoll, Surface Science, vol. 191, p. 585-594, (1987) discloses reflectivity versus angle scans in the Kretschmann ATR configuration of an silver-air interface with p-polarized laser light. If there is within the area of the laser spot on the sample film a step from one thickness to another, two resonance minima are obtained, each at an angle corresponding to one of the two "infinite" layer structures. The intensities of the resonance minima depend on the relative area fraction of the two different interfaces covered by the laser spot.
Rothenhausler and Knoll, Appl. Phys. Lett., vol. 51, p. 783-785, (1987) discloses the use of SPR in combination with diffraction to study a discrete film of two media with different layer structures. The mutual relationship between the reflectance minima of two simultaneous SPR's is used to determine the degree of surface coverage for the respective media.
Yeatman and Ash, SPIE, vol. 897, p. 100-107, (1988) p. 107 discloses a technique for SPR microscopy for studying lateral structure (surface heterogeneity distribution) in biological monolayers and other superimposed layers. It is proposed that the position, width and depth of the reflectance minimum be measured to permit different contrast mechanisms to be separable due to their different relative effects on the three parameters.
Zhang, Y. et al, Surface Sci., vol. 184, p. 214-226, (1987) describes the use of surface plasmon oscillations for the investigations of the kinetics of adsorption of a polystyrene film onto a metallic surface. The resonance angular shift, the halfwidth of the dip and the value of the resonance minimum is used for determining the characteristics of the adsorbed layer.
Silin, V. I., et al., Optics Communications, vol. 97, p. 19-24, (1993) discloses broadening of the surface plasmon line in a biosensor system in which an immunologic reaction of the antigen-antibody type takes place due to irregularities, roughness or even a discrete structure of the studied film, resulting in a sharp decrease of the sensitivity in the measurement of the reflectance curve minimum angle.
Salamon, Z., Wang, Y., Tollin, G. and Macleod, H., Biochimica et Biophysica Acta, 1195(2), p. 267-75 (1994) reports theoretical modelling for experimental surface plasmon resonance results to determine the thickness, refractive index, and extinction coefficient of self-assembled lipid bilayers, and to calculate the adsorbed mass and volume. From this calculated data the average steady state structure of the lipid layer is characterized. It is demonstrated that deposition of a lipid layer may change the SPR-reflectance curve so that (i) the position of the resonance minimum is shifted towards larger incident angles; (ii) the reflected light intensity at the resonance minimum is increased; and (iii) the SPR curve is broadened. The observation of only one resonance minimum indicates that the coverage of the silver surface of the sample molecules is homogeneous.
In summary, while as described above, it has been recognized in the prior art that the shape of the surface plasmon resonance curve may contain certain structural information on an SPR-probed solid or liquid film, measurement of the value (i.e. depth) of the reflectance minimum or the halfwidth of the reflectance curve have never been used in SPR-based biosensor applications for the monitoring of surface interactions. Nor has it been suggested that such reflectance curve information would be of any use therein.